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N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

ABSTRACT: Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F₂), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F₂. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F₂, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F₂ blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury.

SUBMITTER: Lu S

PROVIDER: S-EPMC5216659 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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<i>N</i>-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

Lu Shishi S Zhang Yanmei Y Zhong Shuping S Gao Fenfei F Chen Yicun Y Li Weiqiu W Zheng Fuchun F Shi Ganggang G

Frontiers in pharmacology 20170105

Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthi ...[more]

PMID: 28111550

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Project description:Early growth response-1 (Egr-1), a transcription factor which often underlies the molecular basis of myocardial ischemia/reperfusion (I/R) injury, and oxidative stress, is key to myocardial I/R injury. Silent information regulator of transcription 1(SIRT1) not only interacts with and is inhibited by Egr-1, but also downregulates reactive oxygen species (ROS) via the Forkhead box O1(FOXO1)/manganese superoxide dismutase (Mn-SOD) signaling pathway. N-n-butyl haloperidol iodide (F2), a new patented compound, protects the myocardium against myocardial I/R injury in various animal I/R models in vivo and various heart-derived cell hypoxia/reoxygenation (H/R) models in vitro. In addition, F2 can regulate the abnormal ROS/Egr-1 signaling pathway in cardiac microvascular endothelial cells (CMECs) and H9c2 cells after H/R. We studied whether there is an inverse Egr-1/ROS signaling pathway in H9c2 cells and whether the SIRT1/FOXO1/Mn-SOD signaling pathway mediates this. We verified a ROS/Egr-1 signaling loop in H9c2 cells during H/R and that F2 protects against myocardial H/R injury by affecting SIRT1-related signaling pathways. Knockdown of Egr-1, by siRNA interference, reduced ROS generation, and alleviated oxidative stress injury induced by H/R, as shown by upregulated mitochondrial membrane potential, increased glutathione peroxidase (GSH-px) and total SOD anti-oxidative enzyme activity, and downregulated MDA. Decreases in FOXO1 protein expression and Mn-SOD activity occurred after H/R, but could be blocked by Egr-1 siRNA. F2 treatment attenuated H/R-induced Egr-1 expression, ROS generation and other forms of oxidative stress injury such as MDA, and prevented H/R-induced decreases in FOXO1 and Mn-SOD activity. Nuclear co-localization between Egr-1 and SIRT1 was increased by H/R and decreased by either Egr-1 siRNA or F2. Therefore, our results suggest that Egr-1 inhibits the SIRT1/FOXO1/Mn-SOD antioxidant signaling pathway to increase ROS and perpetuate I/R injury. F2 inhibits induction of Egr-1 by H/R, thereby activating SIRT1/FOXO1/Mn-SOD antioxidant signaling and decreasing H/R-induced ROS, demonstrating an important mechanism by which F2 protects against myocardial H/R injury.

Project description:BackgroundMitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial's structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.Methods and resultsThe H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.ConclusionsDuring myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect.

Project description:BackgroundAfter cerebral injury blood-brain barrier disruption significantly impairs brain homeostasis. Volatile anesthetics have been shown to be protective in ischemia-reperfusion injury scenarios. Their impact on brain endothelial cells after hypoxia-reoxygenation (H/R) has not yet been studied in detail.MethodsRat brain endothelial cells (RBE4) were exposed to severe hypoxia and reoxygenated in air in the presence or absence of sevoflurane. Changes in dextran permeability and architecture of the cellular junctional proteins ZO-1 and ?-catenin were measured. To determine necrosis and apoptosis rate DNA content, LDH release and caspase activity were quantified. The role of vascular endothelial growth factor (VEGF) as an inflammatory mediator increasing vascular permeability was assessed. At the same time, it was evaluated if sevoflurane effects are mediated through VEGF. Results were analyzed by unpaired t-tests or one way-analysis of variance followed by Bonferroni's correction.ResultsH/R led to a 172% increase in permeability (p<0.001), cell swelling and qualitatively but not quantitatively modified expression of ZO-1, ?-catenin and F-actin. In the presence of sevoflurane during reoxygenation, barrier function improved by 96% (p = 0.042) in parallel to a decrease of the cell size and less re-arranged junction proteins and F-actin. Sevoflurane-induced improvement of the barrier function could not be explained on the level of necrosis or apoptosis as they remained unchanged independent of the presence or absence of the volatile anesthetic. Increased expression of VEGF after H/R was attenuated by sevoflurane by 34% (p = 0.004). Barrier protection provided by sevoflurane was similar to the application of a blocking VEGF-antibody. Furthermore, the protective effect of sevoflurane was abolished in the presence of recombinant VEGF.ConclusionsIn H/R-induced rat brain endothelial cell injury sevoflurane maintains endothelial barrier function through downregulation of VEGF, which is a key player not only in mediating injury, but also with regard to the protective effect of sevoflurane.

Dataset Information

N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

Publications

<i>N</i>-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

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